Nerves are composed of motor, sensory and sympathetic components (Evans, G. R. D., Anatomical Record, 2001, 263(4):396-404). Nerves may be designated as primarily motor or sensory; however, no nerve is purely one or the other (Evans, G. R. D., Anatomical Record, 2001, 263(4):396-404). Myelinated and unmyelinated axons comprise the nerve fibers. Motor fibers are primarily myelinated and are outnumbered by unmyelinated sensory fibers 4:1 (Evans, G. R. D., Anatomical Record, 2001, 263(4):396-404). Myelinated fibers range in size from 1-20 μm in diameter while umyelinated fibers are typically below 1 μm in diameter (Carpenter, M. B., Human Neuroanatomy, 1979, p. 71-114 and 188-190; Cajal, S. R., Chapter IX: Nerve Fibers, in Histology of the Nervous System, 1995, p. 209-235).
Nerves carry the peripheral processes or axons of neurons but also consist of Schwann cells and connective tissue sheaths. The most inner sheath, the edoneurium, is composed mainly of longitudinally aligned collagen fibers 30-60 nm in diameter (Carpenter, M. B., Human Neuroanatomy, 1979, p. 71-114 and 188-190; Ross, M. et al., Ch 11: Nervous Tissue, in Histology: A Text and Atlas, 1989, p. 241-281; Peters, A. et al., Ch. 12: Connective Tissue Shealths of Peripheral Nerves, in The Fine Structure of the Nervous System—Neurons and Their Supporting Cells, 1991, p. 384-394). Tiny capillaries (<10 μm), fibroblasts, mast cells and macrophages are also found in the endoneurium. The innermost endoneurial layer is often observed to be in close contact with Schwann cell basal laminae.
The perineurium layer comprises cells that exhibit both myoid and epithelioid features and express basal lamina on both surfaces (Carpenter, M. B., Human Neuroanatomy, 1979, p. 71-114 and 188-190; Ross, M. et al., Ch 11: Nervous Tissue, in Histology: A Text and Atlas, 1989, p. 241-281; Peters, A. et al., Ch. 12. Connective Tissue Shealths of Peripheral Nerves, in The Fine Structure of the Nervous System—Neurons and Their Supporting Cells, 1991, p. 384-394). The cells are interlocked in successive sheets via tight junctions. Blood vessels also infiltrate this layer with the perineurim functioning as a selectively permeable barrier. The outermost perineurial layers are composed of dense concentric layers of mostly longitudinally arranged collagen fibrils ca. 50 nm in diameter with a few fibroblasts and macrophages among the strands.
The outer sheath, the epineurium layer, is a dense collagenous layer surrounding all peripheral nerve trunks (Carpenter, M. B., Human Neuroanatomy, 1979, p. 71-114 and 188-190; Ross, M. et al., Ch 11: Nervous Tissue, in Histology: A Text and Atlas, 1989, p. 241-281; Peters, A. et al., Ch. 12: Connective Tissue Shealths of Peripheral Nerves, in The Fine Structure of the Nervous System—Neurons and Their Supporting Cells, 1991, p. 384-394). Fibers in this layer are disposed mainly longitudinally with diameters between 70-85 nm. Elastin fibers are also present with diameters ranging from 250-500 nm. Fibroblasts and mast cells are scattered throughout this layer.
In most tissues, wound healing is usually a coordinated sequence of events that includes (a) tissue disruption and loss of normal tissue architecture; (b) cell necrosis and hemorrhage; hemostasis (clot formation); (c) infiltration of segmented and mononuclear inflammatory cells, with vascular congestion and tissue edema; (d) dissolution of the clot as well as damaged cells and tissues by mononuclear cells (macrophages); and (e) formation of granulation tissue (fibroplasia and angiogenesis). This sequence of cellular events has been observed in wounds from all tissues and organs generated in a large number of mammalian species (See Berry et al., In: CNS Injuries: Responses and Pharmacological Strategies, 1998, A. Logan and M. Berry, eds., CRC Press, Boca Raton, Fla.; Gailet et al., Curr. Opin. Cell. Biol., 1994, 6:717-725). Therefore, the cellular sequence described above is a stereotype of the repair of all mammalian tissues.
Peripheral nerve injuries are extremely prevalent. Each year, an estimated 50,000 peripheral nerve repair procedures are performed in the United States alone (Evans, G. R. D., Anatomical Record, 2001, 263(4):396-404). Much of what has been learned about peripheral nerve repair has grown out of the treatment of warfare injuries (Neal, N., Military Contributions to Peripheral Nerve Injuries. Gray Matters: Newsletter of the National Capital Neurosurgery Program, 2000: p. 1-5). Unfortunately, despite many advances and creative repair strategies, the functional outcomes of nerve repairs are still far from optimal and motor nerves tend to be more refractory than sensory to full recovery (Evans, G. R. D., Anatomical Record, 2001, 263(4):396-404).
Peripheral nerve injury may result from trauma (e.g., lacerations, gunshot wounds, motor vehicle accidents), acute compression, stretching/tension or disease (e.g., cancer, leprosy). A five category system classifies nerve injuries in terms of increasing severity from first-degree to fifth-degree (Evans, G. R. D., Anatomical Record, 2001, 263(4):396-404). A first-degree injury or neurapraxia involves a temporary conduction block with local demyelination yet complete recovery occurs.
Axotomy (axon severance) occurs after any 2nd degree injury or higher. The more severe traumas often require surgery for a chance for complete or partial recovery. When transected or resected nerve ends can not be coapted without tension, a gap defect results requiring nerve grafting to restore neural continuity (Kline, D., et al., Chapter XXV: Graft Repair, in Atlas of Peripheral Nerve Surgery, 2001, p. 183-187).
Autograft (autologous nerve) is the “gold standard” graft material and is preferentially obtained from harvest of the sural nerve, antebrachial cutaneous radial nerve or superficial sensory radial (SSR) nerve (Kline, D., et al., Chapter XXV: Graft Repair, in Atlas of Peripheral Nerve Surgery, 2001, p. 183-187). The fundamental determinates of functional regeneration for autograft are the endoneurium and remaining Schwann cells since the epineural and perineural elements are trimmed from harvested nerve prior to engraftment.
Although autograft is reported to facilitate neuroregeneration over substantial distances (2-15 cm) (Meek, M. F. and J. H. Coert, Journal of Reconstructive Microsurgery, 2002, 18(2):97-109), it has some disadvantages. Lack of donor supply, donor site morbidity, need for secondary surgical site and insufficient functional outcome are the key disadvantages of autograft repair (Hadlock, T. et al., Archives of Otolaryngology-Head & Neck Surgery, 1998, 124(10):1081-1086; Evans, G. R. D., Anatomical Record, 2001, 263(4):396-404; Meek, M. F. and J. H. Coert, Journal of Reconstructive Microsurgery, 2002, 18(2):97-109). Harvesting donor nerve is also time-consuming and often the fascicles do not match the target nerve in both number and diameter. Central or segmental necrosis can also occur in large diameter grafts (Terzis, J. K. et al., International Angiology, 1995, 14(3):264-277). Thus, there exists a need for neuroregenerative conduits constructed from both natural and synthetic materials to supplant autograft.
Entubulation is the most common alternative to autograft repair (Mackinnon, S. E., Journal of Reconstructive Microsurgery, 2001, 17(8):596-597). In this strategy, severed nerve ends are inserted into the hollow or filled lumen of a biomaterial tube employed to protect, facilitate and guide neuroregeneration. Gaps of centimeters have been regenerated successfully, dependent upon the specific materials employed (Langer, R. and J. P. Vacanti, Tissue Engineering. Science, 1993, 260(5110):920-926; Strauch, B. et al., Journal of Reconstructive Microsurgery, 2001, 17(8):589-595; Meek, M. F. and J. H. Coert, Journal of Reconstructive Microsurgery, 2002; DenDunnen, W. F. A. et al., Microsurgery, 1996, 17(7):348-357; Suzuki, K. et al., Journal of Biomedical Materials Research, 1999, 49(4):528-533; Suzuki, Y. et al., Neuroscience Letters, 1999, 259(2):75-78; Chen, Y. S. et al., Biomaterials, 2000, 21(15):1541-1547; Battiston, B. et al., Microsurgery, 2000, 20(1):32-36; Shen, Z. L. et al., Microsurgery, 2001, 21(1):6-11; Gulati, A. K. et al., Brain Research, 1995, 705(1-2): 118-124; Evans, G. R. D. et al., Biomaterials, 1999, 20(12): 1109-1115).
Vein, denatured muscle, combination vein filled with muscle, silicone, Gore-Tex, and polyglycolic acid (PGA) tubes have been used clinically in humans for nerve reconstruction with success (Meek, M. F. and J. H. Coert, Journal of Reconstructive Microsurgery, 2002). Vein grafts were found suitable for gap lengths of less than 4.5 cm dependent upon the nerve under repair. Muscle grafts appeared suitable for reconstruction of gaps greater than 6 cm in leprosy patients and were judged superior to conventional nerve grafting in repair of 1.5-2.8 cm gaps resulting from laceration injuries. Combination vein filled with muscle conduits have been used to successfully reconstruct 6 cm gaps. The ready supply of vein and muscle make them attractive graft material choices, and combination vein-muscle grafts have shown superior results to vein alone in similar defects.
Hollow GORE-TEX conduits are indicated in reconstructions up to 4 cm and cause less tissue irritation than silicone tubes. Silicone tubes have only shown success for 4 mm gaps and 29% of the tubes needed to be removed due to (compressive) irritation. In clinical studies utilizing PGA tubes, the maximum defect that could be reconstructed was 3 cm and the conduits performed significantly better than autograft. Allografts in combination with systemic immunosuppressive therapy have also been used successfully in the clinic to reconstruct massive (>10-20 cm) peripheral nerve defects although the accompanying therapy is a serious drawback (Mackinnon, S. E. et al., Plastic and Reconstructive Surgery, 2001, 107(6):1419-1429).
Although clinical studies demonstrate that conduits, natural or synthetic, are at least comparable to autograft in repair of short defects (≦ca. 3 cm), there still exists a need for a conduit or scaffold useful for repairing large nerve gaps, such as a bioresorbable synthetic conduit capable of holding permissive tissues.
Biomaterial scaffolds are a fundamental component of tissue reparative, restorative and regenerative strategies, and development of advanced biomaterial scaffolds is crucial to the continued progress and success of the tissue engineered field. Ionically (Ca2+) and covalently (e.g., ethylene diamine) crosslinked alginate foams and gels have been studied for use as tissue scaffolds (Kuo, C. K. and P. X. Ma, Biomaterials, 2001, 22(6):511-521; Suzuki, K. et al., Neuroreport, 1999, 10(14):2891-2894; Suzuki, Y. et al., European Journal of Neuroscience, 2000, 12:287-287). However, these materials have randomly oriented microstructures; therefore, a need exists for imposing structural order on growing/regenerating cells and tissues via scaffold architecture and geometry.
Alginate is a linear polysaccharide discovered by E.C.C. Stanford in 1880 obtained from alkali digestion of various brown sea algae (Schuberth, R. Ionotropic Copper Alginates: Investigations into the formation of capillary gels and filtering properties of the primary membrane, 1992, University of Regensburg: Regensburg; ISP Alginates, Section 3: Algin-Manufacture and Structure, in Alginates: Products for Scientific Water Control, 2000, International Specialty Products: San Diego, pp. 4-7). The polymer chain is composed of β-1→4 linked D-mannuronic acid (M) and α-1→4 linked L-guluronic acid (G) monosaccharides found in three distinct blocks: polyM, polyMG and PolyG blocks. Compositional variation is a reflection of source and processing. The pKa's of the C5 epimers are 3.38 and 3.65 for M and G respectively with the pKa of an entire alginate molecule somewhere in-between (Schuberth, R. Ionotropic Copper Alginates. Investigations into the formation of capillary gels and filtering properties of the primary membrane, 1992, University of Regensburg: Regensburg; ISP Alginates, Section 3: Algin-Manufacture and Structure, in Alginates: Products for Scientific Water Control, 2000, International Specialty Products: San Diego, pp. 4-7).
Alginate forms colloidal gels (high water content gels, hydrogels) with divalent cations. In the alginate ion affinity series Cd2+>Ba2+>Cu2+>Ca2+>Ni2+>Co2+>Mn2+, Ca2+ is the best characterized and most used to form gels (Ouwerx, C. et al., Polymer Gels and Networks, 1998, 6(5):393-408). Studies indicate that Ca-alginate gels form via a cooperative binding of Ca2+ ions by polyG blocks on adjacent polymer chains, the so-called “egg-box” model (ISP Alginates, Section 3: Algin-Manufacture and Structure, in Alginates: Products for Scientific Water Control, 2000, International Specialty Products: San Diego, pp. 4-7). G-rich alginates tend to form thermally stable, strong yet brittle Ca-gels that are likely to undergo syneresis, while M-rich alginates tend to form less thermally stable, weaker but more elastic gels.
Alginate is commercially used as a binding, stabilizing and/or thickening additive in many foods and cosmetics (ISP Alginates, Section 3: Algin-Manufacture and Structure, in Alginates: Products for Scientific Water Control, 2000, International Specialty Products: San Diego, pp. 4-7). Clinically, alginate is used in dental impression materials and hemostatic wound dressings (Blair, S. D. et al., Brit. J. Surg., 1990, 77(5):568-570; Rives, J. M. et al., Calcium alginate versus paraffin gauze in the treatment of scalp graft donor sites, Wounds-a Compendium of Clinical Research and Practice, 1997, 9(6):199-205). Also, alginate-poly-L-lysine encapsulated pancreatic islet cells have been evaluated in a human clinical trial for treatment of type I diabetes (Soon-Shiong, P. Adv. Drug Delivery Reviews, 1999, 35(2-3):259-270; Sandford, P. A. and P. Spoonshiong, Alginate Encapsulation-Update on 1st Human Clinical-Trial with Encapsulated Human Islets in a Type-I-Diabetic Patient with Sustained Islet Function 16 Months Post Encapsulated Islet Transplant, Abstracts of Papers of the American Chemical Society, 1995, 209:44-CELL). Alginate-chitosan PEC beads and films have been made experimentally for cellular immunoprotective capsules and drug release devices (Yan, X. L. et al., Chem. & Pharm. Bull., 2000, 48(7):941-946; Gaserod, O. et al., Biomaterials, 1999, 20(8):773-783; Bartkowiak, K and Hunkeler, D. Annals of the New York Academy of Sciences, 1999, 875:36-45). Ionically (Ca2+) and covalently (e.g., ethylene diamine) crosslinked freeze-dried foams and gels have been studied for use as tissue scaffolds (Kuo, C. K. and P. X. Ma Biomaterials, 2001, 22(6):511-521; Suzuki, K. et al., Neuroreport, 1999, 10(14):2891-2894; Suzuki, Y. et al., Euro. J. Neurosci., 2000, 12:287-287).
Copper capillary alginate gels (CCAG) have been known in the literature for at least 40 years (Schuberth, R., Ionotropic Copper Alginates: Investigations into the formation of capillary gels and filtering properties of the primary membrane. 1992; Thiele, H., Histolyse und Histogenese, Gewebe und ionotrope Gele, Prinzip einer Stukturbildung. 1967). The gels are essentially formed by allowing solutions of Cu2+ to diffuse uniformly into viscous solutions of alginate. During this diffusion process, it is reported that fluid instabilities arise from the friction forces involved in the contraction of alginate polymer chains to the newly forming gel front (Thumbs, J. and H. H. Kohler, Chemical Physics, 1996, 208(1):9-24). Convecting tori, similar to those observed in the Raleigh-Benard model of heat convection, result from these hydrodynamic instabilities. In a sense, these tori tunnels parallel capillaries through the forming gel in the direction of diffusion. A continuous, tubular microstructure is mapped onto the forming gel due to the convective-like process the system undergoes to dissipate energy. Gel capillary diameter can be adjusted by manipulating singly, or in combination, the initial alginate concentration, initial Cu2+ concentration or system pH (Schuberth, R., Ionotropic Copper Alginates: Investigations into the formation of capillary gels and filtering properties of the primary membrane. 1992; Thumbs, J. and H. H. Kohler, Chemical Physics, 1996, 208(1):9-24; Thiele, H., Histolyse und Histogenese, Gewebe und ionotrope Gele, Prinzip einer Stukturbildung. 1967).
However, in common tissue culture media, CCAG alone swells, loses mechanical properties, and eventually dissolve due to a loss of copper ions that are released into the surrounding fluid environment. Accordingly, there is a need for a modified CCAG that provides a stable tissue scaffold in a cell culture environment or within a human or animal.